Excitable Cells - 4

Excitable Cells - 4

1. Ions

   • Charged and moving, ion flow has a current which can be measured
     • A volt meter measures potential difference (p.d.) between 2 points
   • Resistance is the restriction to ion flow
   • Current is the flow of ions i.e. larger pore
     • Less resistance
       • Greater the flow
         • Bigger current
   • Voltage is p.d. -> greater charge differences between 2 areas
     • No p.d.
       • No ion flow
         • Current

2. Electrophysiological recordings

   • Extremely fast events – sub-millisecond timescale upwards
   • Extremely sensitive – as little as one ion channel can be detected
   • Spatial resolution – good
   • Find details of individual channels – activation, inactivation, pore properties

3. Patch clamp recordings of single channels

   • Ion Fluxes (currents) Through Single Channels
   • Glass tube pulled to give very fine tipped (<1μm wide) electrode
     • Pipette (electrode) is filled with electrical conducting solution – pipette solution
       • Pipette is then connected to very fast amplifier and recording equipment when patch clamping in whole brains, proximity to a nerve is detected by changes in electrical resistance

4. Cell-attached configuration

   • To record currents through a limited number (1-2) active channels at cell surface
   • Good for looking at single channel currents in response to regulation of channels by cell
   • Randomly place pipette and if lucky record activity using volt meter

5. Inside- out configuration

   • Use: To record currents through a single active channel away from cell
   • Good for looking at agents that modulate channel by working at its intracellular face

6. Outside-out configuration

   • Use: To record currents through a single active channel away from cell
   • Good for looking at agents that modulate channel by working at its extracellular face

7. Whole-cell configuration - mostly used

   • Use: To record currents through active channels in whole cell
   • Good for looking at cell currents in response to drugs added from outside
   • Good for regulation of channels by cell

8. Single ion channel depends on:

   • How long the channel remains open
   • How big the current is through the channel
   • How often they open and close
   • After the patch clamp = RTP CR
     • clone extracted sequences of amino acids
   • An action potential trace on a graph is composed of voltage across each of the ion channels opening
   • Current (I) = volts (V) X conductance Where V is a constant
     • Implies if we could hold the voltage constant the resulting current would allow us to determine the conductance of the channels